Molecular Biotechnology

, Volume 20, Issue 1, pp 85–93

Aqueous two-phase extraction and purification of animal proteins



Aqueous two-phase systems provide a rapid, easily scalable method for separation of soluble proteins from insoluble materials and other undesired proteins. The method can be operated in continuous mode. It is particularly useful for animal proteins, as it overcomes difficulties of other methods in removing bulk insoluble material, while at the same time providing purification with respect to total soluble protein. This article describes the development of methods for aqueous two-phase extraction and purification of animal proteins, at both laboratory and pilot scale. The strengths, weaknesses, and possible future prospects for the method are discussed.

Index Entries

“Aqueous two-phase” protein enzyme animal purification separation 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Boland, M. J. (1990) Extraction of proteins from animal tissue using multiphase aqueous systems. Bioseparations 1, 293–304.Google Scholar
  2. 2.
    Cole, K. D., Lee, T. K., and Lubon, H. (1997) Aqueous two-phase partitioning of milk proteins. Application to human protein C secreted in pig milk. Appl. Biochem. Biotechnol. 1, 97–112.Google Scholar
  3. 3.
    Harris, D. P., Andrews, A. T., Wright, D., Pyle, D. L., and Asenjo, J. A. (1997) The application of aqueous two-phase systems to the purification of pharmaceutical proteins from transgenic sheep milk. Bioseparation 7, 31–37.PubMedCrossRefGoogle Scholar
  4. 4.
    Ollero, M., Garcia-Lopez, N., Perez-Re, R., Cebrian-Perez, J. A., and Muino-Blanco, T. (1997) Surface changes of ram spermatozoa by adsorption of homologous and heterologous seminal plasma proteins revealed by partition in an aqueous two-phase system. Reprod. Fertil. Dev. 9, 381–390.PubMedCrossRefGoogle Scholar
  5. 5.
    Perez, M. T., Garcia-Perez, A. I., Lucas, L., and Sancho, P. (1996) A single partitioning step in aqueous polymer two-phase systems reduces hypnotized rat erthrocyte heterogeneity. J. Chromatog. B. Biomed. Appl. 17, 680 183–188.CrossRefGoogle Scholar
  6. 6.
    Fritsch, M., Anderson, I. Leary, C. M., and Gorski J (1997) Two populations of the estrogen receptor separated and characterized using aqueous two-phase partitioning. Biochemistry 20, 6230–6242.CrossRefGoogle Scholar
  7. 7.
    Walker, S. G., Dale, C. J., and Lyddiatt, A. (1996) Aqueous two-phase partitioning of complex protein feedstocks derived from brain tissue homogenates. J. Chromatog. B. Biomed. Appl. 17, 680 91–96.CrossRefGoogle Scholar
  8. 8.
    Tolstoguzov, V. (2000) Compositions and phase diagrams for aqueous systems based on proteins and polysaccharides. Int. Rev. Cytol. 192, 3–31.PubMedCrossRefGoogle Scholar
  9. 9.
    Johansson, G. and Walter, H. (2000) Partitioning and concentrating biomaterials in aqueous phase systems. Int. Rev. Cytol. 192, 33–60.PubMedGoogle Scholar
  10. 10.
    Gehrke, S. H., Vaid, N. R., and McBride J. F. (1998) Protein sorption and recovery by hydrogels using principles of aqueous two-phase extraction. Biotechnol. Bioeng. 20, 416–427.CrossRefGoogle Scholar
  11. 11.
    Johansson, G. and Kopperschlager, G. (1987) Effects of organic solvents on the partitioning of enzymes in aqueous two-phase systems. J. Chromatogr. 388, 295–305.PubMedCrossRefGoogle Scholar
  12. 12.
    Boland, M. J., Hesselink, P. G. M., and Hustedt, H. (1989) Extractive purification of enzymes from animal tissue using aqueous phase systems. J. Biotechnol. 11, 337–352.CrossRefGoogle Scholar
  13. 13.
    Tjerneld, F., Berner, S., Cajarville, A., and Johansson, G. (1986) New aqueous two-phase system based on hydroxypropyl starch useful for enzyme purification. Enzyme Microb. Technol. 8, 417–423.CrossRefGoogle Scholar
  14. 14.
    Alves, J. G., Chumpitaz, L. D., da Silva, L. H., Franco, T. T., and Meirelles, A. J. (2000) J. Chromatogr. B. Biomed. Sci. Appl. 23, 235–239.CrossRefGoogle Scholar
  15. 15.
    Johansson, G. and Joelsson, M. (1987) Effect of polymer structure on affinity partitioning of lactate dehydrogenase in polymer-water two-phase systems. J. Chromatog. 411, 161–166.CrossRefGoogle Scholar
  16. 16.
    Johansson, G. and Joelsson, M. (1986) Liquid-liquid extraction of lactate dehydrogenase from muscle using polymer-bound triazine dyes. Applied Biochem Biotechnol. 13, 15–27.Google Scholar
  17. 17.
    Tjerneld, F., Johansson, G., and Joelsson, M. (1987) Affinity liquid-liquid extraction of lactate dehydrogenase on a large scale Biotechnol. Bioeng. 30, 809–816.CrossRefPubMedGoogle Scholar
  18. 18.
    Joelsson, M. and Johansson, G. (1987) Sequential liquid-liquid extraction of some enzymes from porcine muscle using polymer-bound triazine dyes. Enzyme Microb. Technol. 9, 233–237.CrossRefGoogle Scholar
  19. 19.
    Shibusawa, Y., Kihira, S., and Ito, Y. (1998) One-step purification of proteins from chicken egg white using counter-current chromatography. J. Chromatog. B. Biomed. Sci. Appl. 29, 301–305.CrossRefGoogle Scholar
  20. 20.
    Boland, M. J., Hesselink, P. G. M., Papamichael, N., and Hustedt, H. (1991) Extractive purification of enzymes from animal tissue using aqueous two-phase systems: pilot scale studies. J. Biotechnol. 19, 19–34.PubMedCrossRefGoogle Scholar
  21. 21.
    Johansson, G. (1996) Aqueous two-phase systems with a liquid protein (bovine serum albumin) phase for partitioning of enzymes J. Chromatog. B. Biomed. Appl. 17, 123–130.CrossRefGoogle Scholar
  22. 22.
    Johansson, H. O., Persson, J., and Tjerneld, F. (1999) Thermoseparating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification. Biotechnol. Bioeng. 66, 247–257.PubMedCrossRefGoogle Scholar
  23. 23.
    Kopperschlager, G. (2000) Effects of specific binding reactions on the partitioning behavior of biomaterials. Int. Rev. Cytol. 192, 61–97.PubMedGoogle Scholar
  24. 24.
    Hustedt, H., Börner, B., Kroner, K.-H., and Papamichael, N. (1987) Fully automated continuous crosscurrent extraction of enzymes in a two-stage plant. Biotechnol. Tech. 1, 49–54.CrossRefGoogle Scholar
  25. 25.
    Hustedt, H., Kroner, K.-H., and Papamichael, N. (1988) Continuous cross-current aqueous two-phase extraction of enzymes from biomass: Automated recovery in production scale. Process Biochemistry, October 1988 129–137.Google Scholar
  26. 26.
    Hustedt, H., Schutte, H., Paulssen, J., and Boland, M. J. (1990) Protein purification by two-step extraction followed by hydrophobic interaction chromatography, in Fermentation Technologies-industrial applications (Yu, P.-L., ed.), Elsevier Applied Science (London and New York), pp. 439–444.Google Scholar
  27. 27.
    Schutte, H., Papamichael, N., Paulsen, J., and Hustedt, H. (1991) Integration of hydrophobic adsorption into protein purification schemes, in Fundamentals of Adsorption (3rd international conference) Mersmann, A. B. and Scholl, S. E., eds. Engineering Foundation (New York), pp. 795–803.Google Scholar
  28. 28.
    Kroner, K.-H., Hustedt, H., and Kula, M.-R. (1984) Extractive enzyme recovery: economic considerations. Process Biochemistry 19, 170–179.Google Scholar
  29. 29.
    Hustedt, H. (1986) Extractive enzyme recovery with simple recycling of phase forming chemicals. Biotechnol. Lett. 8, 791–796.CrossRefGoogle Scholar
  30. 30.
    Vernan, J. and Kula, M.-R. (1989) Extraction of proteins from biological raw material using aqueous PEG-citrate phase systems. Lecture presented to the 6th International conference on phase partitioning, Assmanshausen, Germany.Google Scholar

Copyright information

© Humana Press Inc 2002

Authors and Affiliations

  1. 1.New Zealand Dairy Research InstitutePalmerston NorthNew Zealand

Personalised recommendations